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Wildtype One @wildtypeone.bsky.social · 6d

👌

Reposted by Wildtype One

Victor Tatarskiy @greenrat.bsky.social · 6d

Neat trick if you polycolonal ab's suck. Incubate them with fixed cells with a KO of your protein of interest, then spin. Protocol here: www.med.upenn.edu/markslab/ass...
I was amazed how well it worked on first try (I'm sure that I can completely eliminate unspecific bands)
#WesternBlot #cellsky

On the left - western blot of B16F10 cells wt and KO for CDK8. Our in house produced antibodies give a lot of unspecific bands. On the right same probes with antibodies preincubated with fixed CDK8 KO cells - there is a specific band and faint unspecific bands, which can be probably eliminated with increase of amount of KO cells.

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Wildtype One @wildtypeone.bsky.social · 6d

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Wildtype One @wildtypeone.bsky.social · 6d

✅ What to do instead

• Keep detector sensitivity high; ensure autofluorescence is above noise
• Stay in the linear range; adjust voltages so the brightest fluorochromes don’t saturate
• If autofluorescence swamps a channel, move to lower-background dyes (red)

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Wildtype One @wildtypeone.bsky.social · 6d

❌ Wrong move.

• Autofluorescence is a cell-intrinsic property, not just noise
• Reducing sensitivity hides autofluorescence and your real signals
• The solution is experimental design—choosing fluorochromes and channels that separate signal from background

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Wildtype One @wildtypeone.bsky.social · 6d

Most unstained cells glow, especially under UV, violet, or blue lasers. This autofluorescence can overlap with weak signals — which is why many users see it as a nuisance.

The common bad advice? Crank detector sensitivity way down so negative controls look dark.

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Wildtype One @wildtypeone.bsky.social · 6d

Turning down the TV brightness won't remove static. It just makes everything else harder to see.

Lowering detector sensitivity in flow cytometry is the same. Instead, tune the “channel” by picking fluorochromes that separate cleanly from autofluorescence.

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Wildtype One @wildtypeone.bsky.social · 6d

Here's a very bad flow cytometry advice: “autofluorescence needs to be minimized”

Analogy: Imagine you're watching TV and you have static...

(a thread)

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Wildtype One @wildtypeone.bsky.social · 10d

Takeaway:

1. Don’t sink weeks into a fragile assay
2. Use high-throughput assays to discover
3. Use Western blots to validate the discovery

— Wildtype One 🧬

Wildtype One @wildtypeone.bsky.social · 10d

He’d be the one in lab meetings asking:

🤔“Why do we accept Western blots failing half the time as normal science?”

Instead:

👉 He’d push multiplex assays and large proteomics
👉 He'd want to get a global view of cell signaling before looking at a single protein

(3/4)

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Wildtype One @wildtypeone.bsky.social · 10d

He probably never did one, nor was he a biologist...

But Einstein hated finicky, low-reproducibility work.

He criticized “stamp collecting”

i.e., piling up empirical details without deeper explanatory power.

(2/4)

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Wildtype One @wildtypeone.bsky.social · 10d

Albert Einstein would've hated Western blots...

(a thread)

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Reposted by Wildtype One

Christophe 🔬 L @christlet.bsky.social · 15d

Historians of science agree that the postdoc was invented just after the Western blot, as even the inventor wouldn't want to run one

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Wildtype One @wildtypeone.bsky.social · 13d

tweet of the day

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Wildtype One @wildtypeone.bsky.social · 13d

🧫 Join 400+ elite researchers getting weekly lab hacks with our newsletter (it’s free) 👉 wildtypeone.substack.com/about

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Wildtype One @wildtypeone.bsky.social · 13d

5️⃣ Tukey’s deeper point

Biology runs on plasticity, degeneracy, and context.

Your job isn’t to oversimplify. It’s to frame questions that cut into the causal architecture—even if the answers are noisy, probabilistic, or incomplete.

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Wildtype One @wildtypeone.bsky.social · 13d

4️⃣ Why this matters for a young researcher

Because they're pressured to publish “clean data with tight error bars.”

The danger here is mistaking polish for relevance.

Approximate maps of meaningful processes outlast perfect dissections of trivial ones.

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Wildtype One @wildtypeone.bsky.social · 13d

Example B - Right question, approximate answer: “Does inhibiting KRAS rewire in vivo signaling networks?” --> Answered imperfectly with spatial transcriptomics or lineage tracing, but relevant.

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Wildtype One @wildtypeone.bsky.social · 13d

3️⃣ Examples from cancer research

Example A - Wrong question, exact answer: “What’s the EC₅₀ of this KRAS inhibitor in one colorectal cell line?” --> Easy, precise, but tells you little about patient resistance.

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Wildtype One @wildtypeone.bsky.social · 13d

🥐🧈 It’s like having a precise amount of high-quality butter, but forgetting what dish you're cooking.

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Wildtype One @wildtypeone.bsky.social · 13d

2️⃣ Precision ≠ truth in living systems

Physics rewards exactness. Biology punishes it.

Transcript counts, single-cell trajectories, and proteomic hits are statistical shadows of moving targets.

A p-value to five decimals is meaningless without a good context.

For example...

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Wildtype One @wildtypeone.bsky.social · 13d

The harder, “right” question might be: “Does this perturbation rewire lineage commitment in a way that sustains heterogeneity?”

In this case, your answer will be approximate, but biologically richer.

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Wildtype One @wildtypeone.bsky.social · 13d

1️⃣ The right question is messy

A KRAS mutation means one thing in a 2D cell line, another in a hypoxic tumor niche.

So if you chase the exact fold-change of protein X after drug Y in 21% O₂, you’ll get pristine, reproducible data… that says little about real tumors.

[...]

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Wildtype One @wildtypeone.bsky.social · 13d

This quote from the famous statistician, John Tukey, had 5 warnings to biologists 🧫

(a thread)

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Wildtype One @wildtypeone.bsky.social · 15d

Einstein wasn't a biologist.

But imagine he's your colleague today: What would your lab look like? How would your experiments change?

We answer that question in today's Elite Researcher Weekly (in 3 min or less) 👇

If Einstein designed your experiments in 2025…

“If you can’t explain it to a six-year-old, you don’t understand it yourself.” — Albert Einstein Dear Elite Researcher, Einstein was NOT a wizard with his hands.

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